C Introduction In vivo oral absorption & toxicity 0 . 4 # # # in vivo ... Nanotox congres... · C a...
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Caecum Small Int PP MLN Liver Kidneys Spleen 0.0 0.1 0.2 0.3 0.4 0.5 2 4 6 Ce (ppm) Vehicle - PEG CeO 2 Citrate CeO 2 - PEG Vehicle - Citrate ## # ** ** * * * * *** ** ** ** # ** ** Cabellos J 1 , Gimeno I 1 , Damásio J 1 , Aimonen K 2 , Lindberg HK 2 , Vales G 2 , Alturi R 3 , Jensen KA 3 , Vázquez-Campos S 1 , Catalán J 2 , Janer G 1 1 Leitat Technological Center, Terrassa (Barcelona), Spain. email: [email protected] 2 Finnish Institute of Occupational Health, Helsinki, Finland 3 National Research Centre for the Working Environment, Copenhagen, Denmark Role of composition, coating and specific surface area on the biokinetics and toxicity of orally administered nanomaterials . Introduction There is limited understanding on how different physicochemical properties affect NM toxicity, especially by routes other than inhalation. By means of in vitro (differentiated Caco-2 cell model) and in vivo (acute & subacute studies in rodents) assays, we investigated the role of different NM properties (size, coating and surface area) on systemic absorption and on toxicity by the oral route. Materials and Methods NP characterization . Conclusions Cell barrier translocation was very low in vitro (only sporadically detected for the smallest TiO 2 NP). In spite of the lack of in vitro intestinal absorption, in vivo results suggest that some absorption or TiO 2 and CeO 2 NPs occurs after dose-repeated administration of. The Caco-2 monolayer system does not cover all mechanisms of intestinal NP translocation. In vivo repeated-dose oral gavage of TiO 2 (E171) study indicates very low influence of fasting on intestinal absorption of TiO 2 NPs. Coating hydrophobicity influenced the intestinal absorption of CeO 2 NPs in the in vivo repeated-dose oral study. Short-term oral dose of SiO 2 NPs with different specific surface properties did not show major toxicological effects up to 1000 mg/kg b.w. www.guidenano.eu In vitro assays in Caco-2 cells (TiO 2 & CeO 2 NPs) 14-day oral dose-repeated administration of TiO 2 (E171) & CeO 2 NPs in rats Daily Oral gavage -- 14 days In vitro oral absorption & toxicity Membrane integrity measured by % Lucifer Yellow permeability and transepithelial electric resistance (TEER) was not affected by any of the TiO 2 or CeO 2 NPs up to 48h of exposure. NPs could move through the insert in the absence of cells, but insert permeability was decreased when big aggregates were formed (as in uncoated CeO 2 NP), or for highly hydrophobic particles (CeO 2 -DDPA). For TiO 2 NPs, only TiO 2 (1) NPs showed detectable levels in the basolateral side. No translocation of Ce 2+ through the Caco-2 cell barrier was detected for any of the CeO 2 NPs. TiO 2 _E171 CeO 2 – Cit CeO 2 -PEG Table 1. Percentage of the applied Ti or Ce 2+ that is present in the basal compartment after 48 h of exposure to TiO 2 and CeO 2 NPs. Results are means of 3 independent assays. Ti 2+ LOD= 0.072 μg/mL for TiO 2 (2)_E171; Ce 2+ LOD = 0.004 μg/mL. In vivo oral absorption & toxicity Body Weight and Clinical signs of toxicity Gross pathology & organ weight Small intestine histopathology Ti /Ce levels by ICP-MS Liver, spleen, small intestine, Peyer’s patches, caecum, mesenteric lymph nodes, lungs, epididymis, testes. TEM evaluations : Small intestine and Peyer’s Patches) www.nanocalibrate.eu NP: Non-porous ; P: Porous SiO 2 NPs: Synthesized by NRCWE (Denmark). TiO 2 (1) TiO 2 (2) E171 CeO 2 - Uncoated CeO 2 - DDPA CeO 2 -Citrate CeO 2 -PEG Size range by TEM (nm) 18 ± 8 117 ± 54 4-8 4-8 4 - 8 4 - 8 DLS Z-Avg 229 249 1056 17 147 81 131 529 PDI 0,408 0,097 0,719 0,386 0,303 0,374 0,252 0,350 Conc (mg/mL) 0,1 0,1 0,1 0,1 0,1 10 0,1 10 Dispersion media DMEM Tap water DMEM DMEM DMEM MiliQ water MiliQ water DMEM 24 h 48 h exposure 21 days cell differentiation in inserts Sampling of basal compartment for ICP-MS analyses Membrane integrity TEER measurement Lucifer Yellow assay All nanoparticles were dosed at [100 μg/mL] as TiO 2 or CeO 2 except for CeO 2 -DDPA [50μg/mL] as CeO 2 Transwell Ø 3 μm Dispersion method: TiO 2 (1) & TiO 2 (2) E171: Sonication in DMEM. Tip 6 mm Ø, 20% amplitude, 15 min. CeO 2 NPs: Dilution in DMEM from stock solution and vortexing Dispersion method: TiO 2 (E171): sonication in water (6 mm Ø, 20% ampl., 15 min) CeO 2 NPs: Dilution in water from stock solution and vortexing * Non-fasting vs. Fasting (2h) conditions were evaluated for E171 * 100 mg/kg b.w. (p.o.) as TiO 2 120 mg/kg b.w. (p.o.) as CeO 2 Short-term oral dose administration of SiO 2 NPs in mice Daily Oral gavage 5 days SiO 2 _100NP SiO 2 _100P SiO 2 _300NP SiO 2 _300P Gross pathology & organ weight Small intestine histopathology Small intestine genotoxicity (Comet assay) TNF- levels in intestinal mucosa Haematology and blood chemistry (AST, ALT, creatinine, total protein content) Systemic genotoxicity (Frequency of MNs in normochromatic (NCE) & polychromatic erythrocytes (PCE)) 100 & 1000 mg/kg b.w. (p.o.) as SiO 2 3 weeks recovery Body Weight and Clinical signs of toxicity Day 6 Day 26 Dispersion method : Tip sonication 3 mm Ø, 20% amplitude, 5 min (except SiO 2 _100P, 10 min) . Dispersant : CMC- Tween (0.2% w/v 1% (v/v) Ti 2+ and Ce 2+ content by ICP-MS TiO 2 _(1) TiO 2 (2) E171 CeO 2 -Unc CeO 2 - CIT CeO 2 - PEG CeO 2 - DDPA Insert without cell membrane [%) 52,6 82,5 8,86 70,6 53,75 0,19 Insert with cell membrane [μg/mL) 0,30 < LOD <LOD <LOD <LOD <LOD No clinical signs of toxicity / No differences in body weight / No histopathological findings in small intestine Figure 1. Titanium levels in tissues of animals orally administered the vehicle or TiO 2 NPs. Mean ± SEM. T-test with Welch correction (*) p value <0.05 (**) <0.01 TiO 2 treatment vs. vehicle. T-test with Welch correction p value (#) <0.05 Fasted vs. non-Fasted. PP: Peyer’s patch; MLN: Mesenteric lymph nodes. 14-day oral dose-repeated administration of TiO 2 (E171) & CeO 2 NPs in rats Kidneys Caecum Spleen Liver Epididymis Small Int PP MLN Lungs Testes 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Ti (ppm) Vehicle - Non fasted Vehicle - Fasted E171 - Non Fasted E171- Fasted * # # ** ** * Figure 2. Cerium levels in tissues of animals orally administered the vehicle or the cerium oxide nanoparticles. Mean ± SEM. T-test with Welch correction p value (*) <0.05 (**) <0.01 CeO 2 treatment vs. corresponding vehicle. T-test with Welch correction P value # <0.05 ## <0.01 CeO 2 -Citrate vs CeO 2 -PEG. Short-term oral dose administration of SiO 2 NPs in mice TiO 2 NPs: Ti levels higher than controls in caecum, small intestine (non-fasted), and liver (fasted). CeO 2 NPs: Ce levels higher than controls in caecum, small intestine, Peyer’s patches, liver, kidneys & spleen. Cerium levels in liver, kidneys and spleen account for 0.0004% and 0.0002% of the total administered cerium for CeO 2 -CIT and CeO 2 -PEG, respectively. CTRL MMS 100NP LD 100NP HD 100P LD 100P HD 300NP LD 300NP HD 300P LD 300P HD 0 20 40 60 80 100 Comet Tail Intensity (%) *** Day 6 CTRL MMS 100NP LD 100NP HD 100P LD 100P HD 300NP LD 300NP HD 300P LD 300P HD 0 20 40 60 80 100 Comet Tail Intensity (%) *** Day 26 No clinical signs of toxicity. No differences in body weight. Minor histopathological findings in small intestine (see figure 3) of slight higher incidence in animals administered with SiO 2 _100P , low dose at day 6. Some statistical significant effects on organ weight: Decrease in spleen weight: SiO 2 _100NP; high dose; Day 6 (0.35 % vs. 0.43 % b.w. in vehicle control). Increase in liver weigh: SiO 2 _300P, high dose, Day 26 (5.7 % vs. 5.0 % b.w. in vehicle control) and increase in spleen weight (0.49 % vs. 0.36 % b.w. in vehicle control). Some haematological and blood biochemistry differences of unclear relevance: Increase in MPV, PDWsd and PDWcv [SiO 2 _300 NP, low dose, Day 6] No significant effects in the percentage of Comet tail intensity in intestinal cells (see figure 4). No significant effects in TNF- levels in intestinal mucosa. No significant differences in the frequency of MNs in erythrocytes at day 6 or 26. Figure 4. DNA tail intensity (%) of jejunum samples from animals orally administered the vehicle (CTRL), the positive control MMS [100 mg/kg b.w.] or SiO 2 nanoparticles. Mean of medias ± SEM (n=6). One-way ANOVA followed by Dunnett’s test (***) <0.01. “This project has received funding from the European Union’s Seventh Programme for research, technological development and demonstration under grant agreements No 604387(GUIDEnano) and No 686239 (caLIBRAte)” SiO 2 _100 NP SiO 2 _100 P SiO 2 _300 NP SiO 2 _300 P Size range by TEM (nm) 114 ± 31 126 ± 10 346 ± 18 291 ± 54 Specific surface area (m 2 /g) 22 844 10 828 TiO 2 (1): Provided by L’Urederra (Spain) under the framework of Nanopolytox project (Grant agreement No.247899). TiO 2 (2) E171: Food grade pigment provided by Sensient Colors S.A. (México) CeO 2 NPs: Synthesized by Plasmachem GmbH (Germany) Figure 3. Example images for SiO 2 _100 NP, low dose (A) Small intestine: Presence of some necrotic/apoptotic cells in the lamina propria (white arrows). (B) Some vacuolated cells, possibly macrophages on the tip of the villi. 50 μm (A) 50 μm (B)
Transcript of C Introduction In vivo oral absorption & toxicity 0 . 4 # # # in vivo ... Nanotox congres... · C a...
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Cabellos J1, Gimeno I1, Damásio J1, Aimonen K2, Lindberg HK2, Vales G2, Alturi R3, Jensen KA3, Vázquez-Campos S1, Catalán J2, Janer G1
1Leitat Technological Center, Terrassa (Barcelona), Spain. email: [email protected] 2Finnish Institute of Occupational Health, Helsinki, Finland 3National Research Centre for the Working Environment, Copenhagen, Denmark
Role of composition, coating and specific surface area on the biokinetics and toxicity of orally administered nanomaterials
. Introduction
There is limited understanding on how different physicochemical properties affect NM toxicity, especially by routes other than inhalation. By means of in vitro (differentiated Caco-2 cell model) and in vivo (acute & subacute studies in rodents) assays, we investigated the role of different NM properties (size, coating and surface area) on systemic absorption and on toxicity by the oral route.
Materials and Methods
NP characterization
.
Conclusions Cell barrier translocation was very low in vitro (only sporadically detected for the smallest TiO2 NP).
In spite of the lack of in vitro intestinal absorption, in vivo results suggest that some absorption or TiO2 and CeO2 NPs occurs after dose-repeated administration of. The Caco-2 monolayer system does not cover all mechanisms of intestinal NP translocation.
In vivo repeated-dose oral gavage of TiO2 (E171) study indicates very low influence of fasting on intestinal absorption of TiO2 NPs.
Coating hydrophobicity influenced the intestinal absorption of CeO2 NPs in the in vivo repeated-dose oral study.
Short-term oral dose of SiO2 NPs with different specific surface properties did not show major toxicological effects up to 1000 mg/kg b.w.
www.guidenano.eu
In vitro assays in Caco-2 cells (TiO2 & CeO2 NPs)
14-day oral dose-repeated administration of TiO2 (E171) & CeO2 NPs in rats
Daily Oral gavage -- 14 days
In vitro oral absorption & toxicity
Membrane integrity measured by % Lucifer Yellow permeability and transepithelial electric
resistance (TEER) was not affected by any of the TiO2 or CeO2 NPs up to 48h of exposure.
NPs could move through the insert in the absence of cells, but insert permeability was
decreased when big aggregates were formed (as in uncoated CeO2 NP), or for highly hydrophobic
particles (CeO2-DDPA).
For TiO2 NPs, only TiO2 (1) NPs showed detectable levels in the basolateral side.
No translocation of Ce2+ through the Caco-2 cell barrier was detected for any of the CeO2 NPs.
TiO2_E171
CeO2 – Cit
CeO2-PEG
Table 1. Percentage of the applied Ti or Ce2+ that is present in the basal compartment after 48 h of exposure to TiO2 and CeO2 NPs. Results are means of 3 independent assays. Ti2+ LOD= 0.072 µg/mL for TiO2(2)_E171; Ce2+ LOD = 0.004 µg/mL.
In vivo oral absorption & toxicity
Body Weight and Clinical signs of toxicity
Gross pathology & organ weight
Small intestine histopathology
Ti /Ce levels by ICP-MS
Liver, spleen, small intestine, Peyer’s patches, caecum, mesenteric lymph nodes, lungs, epididymis, testes. TEM evaluations : Small intestine and Peyer’s Patches)
www.nanocalibrate.eu
NP: Non-porous ; P: Porous SiO2 NPs: Synthesized by NRCWE (Denmark).
TiO2(1) TiO2 (2)
E171 CeO2 -
Uncoated CeO2- DDPA CeO2-Citrate CeO2-PEG
Size range by TEM (nm)
18 ± 8 117 ± 54 4-8 4-8 4 - 8 4 - 8
DLS
Z-Avg 229 249 1056
17 147 81 131 529
PDI 0,408 0,097 0,719 0,386 0,303 0,374 0,252 0,350
Conc (mg/mL)
0,1 0,1 0,1 0,1 0,1 10 0,1 10
Dispersion media
DMEM Tap water DMEM DMEM DMEM MiliQ water
MiliQ water
DMEM
24 h 48 h
exposure
Seeding of Caco2 cells Dispersion in cell
culture medium
21d 48h
Sampling for ICP-MS analyses
• Dialysis
•Acid dgestion
• ICP-MS Ti levels
Calibration
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24h
Membrane
integrity
• Lucifer yellow
100 µg/mL
21 days cell differentiation in inserts
Sampling of basal compartment for ICP-MS analyses
Membrane integrity TEER measurement Lucifer Yellow assay
All nanoparticles were dosed at [100 µg/mL] as TiO2 or CeO2 except for CeO2 -DDPA [50µg/mL] as CeO2
Transwell Ø 3 µm
Dispersion method: TiO2 (1) & TiO2 (2) E171: Sonication in DMEM. Tip 6 mm Ø, 20% amplitude, 15 min. CeO2 NPs: Dilution in DMEM from stock solution and vortexing
Dispersion method: TiO2 (E171): sonication in water (6 mm Ø, 20% ampl., 15 min) CeO2 NPs: Dilution in water from stock solution and vortexing * Non-fasting vs. Fasting (2h) conditions were evaluated for E171
* 100 mg/kg b.w. (p.o.) as TiO2 120 mg/kg b.w. (p.o.) as CeO2
Short-term oral dose administration of SiO2 NPs in mice
Daily Oral gavage 5 days
SiO2_100NP
SiO2_100P
SiO2_300NP
SiO2_300P
Gross pathology & organ weight
Small intestine histopathology Small intestine genotoxicity (Comet assay)
TNF- levels in intestinal mucosa
Haematology and blood chemistry (AST, ALT, creatinine, total protein content)
Systemic genotoxicity (Frequency of MNs in normochromatic (NCE) & polychromatic erythrocytes (PCE))
100 & 1000 mg/kg b.w. (p.o.) as SiO2
3 weeks recovery
Body Weight and Clinical signs of toxicity
Day 6 Day 26
Dispersion method: Tip sonication 3 mm Ø, 20% amplitude, 5 min (except SiO2_100P, 10 min) . Dispersant : CMC-Tween (0.2% w/v 1% (v/v)
Ti2+ and Ce2+ content by ICP-MS
TiO2_(1) TiO2 (2) E171 CeO2-Unc CeO2 - CIT CeO2- PEG CeO2 - DDPA
Insert without cell membrane [%)
52,6 82,5 8,86 70,6 53,75 0,19
Insert with cell membrane [µg/mL)
0,30 < LOD <LOD <LOD <LOD <LOD
No clinical signs of toxicity / No differences in body weight / No histopathological findings in small intestine
Figure 1. Titanium levels in tissues of animals orally administered the vehicle or TiO2 NPs. Mean ± SEM. T-test with Welch correction (*) p value <0.05 (**) <0.01 TiO2 treatment vs. vehicle. T-test with Welch correction p value (#) <0.05 Fasted vs. non-Fasted. PP: Peyer’s patch; MLN: Mesenteric lymph nodes.
14-day oral dose-repeated administration of TiO2(E171) & CeO2 NPs in rats
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Figure 2. Cerium levels in tissues of animals orally administered the vehicle or the cerium oxide nanoparticles. Mean ± SEM. T-test with Welch correction p value (*) <0.05 (**) <0.01 CeO2 treatment vs. corresponding vehicle. T-test with Welch correction P value # <0.05 ## <0.01 CeO2-Citrate vs CeO2-PEG.
Short-term oral dose administration of SiO2 NPs in mice
TiO2 NPs: Ti levels higher than controls in caecum, small intestine (non-fasted), and liver (fasted). CeO2 NPs: Ce levels higher than controls in caecum, small intestine, Peyer’s patches, liver, kidneys & spleen. Cerium levels in liver, kidneys and spleen account for 0.0004% and 0.0002% of the total administered cerium for CeO2-CIT and CeO2-PEG, respectively.
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No clinical signs of toxicity.
No differences in body weight.
Minor histopathological findings in small intestine (see figure 3) of slight higher incidence in
animals administered with SiO2_100P , low dose at day 6.
Some statistical significant effects on organ weight:
Decrease in spleen weight: SiO2_100NP; high dose; Day 6 (0.35 % vs. 0.43 % b.w. in vehicle control).
Increase in liver weigh: SiO2_300P, high dose, Day 26 (5.7 % vs. 5.0 % b.w. in vehicle control) and increase in
spleen weight (0.49 % vs. 0.36 % b.w. in vehicle control).
Some haematological and blood biochemistry differences of unclear relevance:
Increase in MPV, PDWsd and PDWcv [SiO2_300 NP, low dose, Day 6]
No significant effects in the percentage of Comet tail intensity in intestinal cells (see figure 4).
No significant effects in TNF- levels in intestinal mucosa.
No significant differences in the frequency of MNs in erythrocytes at day 6 or 26.
Figure 4. DNA tail intensity (%) of jejunum samples from animals orally administered the vehicle (CTRL), the positive control MMS [100 mg/kg b.w.] or SiO2 nanoparticles. Mean of medias ± SEM (n=6). One-way ANOVA followed by Dunnett’s test (***) <0.01.
“This project has received funding from the European Union’s Seventh Programme
for research, technological development and demonstration under grant agreements No 604387(GUIDEnano) and No 686239 (caLIBRAte)”
SiO2_100 NP SiO2_100 P SiO2_300 NP SiO2_300 P
Size range by TEM (nm) 114 ± 31 126 ± 10 346 ± 18 291 ± 54
Specific surface area (m2/g) 22 844 10 828
TiO2(1): Provided by L’Urederra (Spain) under the framework of Nanopolytox project (Grant agreement No.247899). TiO2(2) E171: Food grade pigment provided by Sensient Colors S.A. (México) CeO2 NPs: Synthesized by Plasmachem GmbH (Germany)
Figure 3. Example images for SiO2_100 NP, low dose (A) Small intestine: Presence of some necrotic/apoptotic cells in the lamina propria (white arrows). (B) Some vacuolated cells, possibly macrophages on the tip of the villi. 50 µm
(A)
50 µm
(B)
